The V3 domain of HIV-1 gp120 has been identified as the principal neutralizing domain for T cell-tropic HIV-1 isolates, but many anti-V3 antibodies ha lower potencies for macrophage-tropic, primary isolates. We have recently found that V1/V2 domain of HIV-1 gp120 contains highly conserved epitopes that can mediate potent neutralization of macrophage- tropic primary HIV-1 isolates. Although the V1/V2 domain is one of the most variable regions of HIV env, the V2 region contain invariant residues which apparently are part of highly conserved neutralization epitopes. These are conformation in nature, and in some cases, glycan-dependent, and thus can not be expressed as peptide. We have developed a fusion glycoprotein that effectively expresses the correctly folded and glycosylated V1/V2 and V3 domains of gp120 in the absence of other HIV sequences. Immunization with these proteins produces antibodies with potent neutralizing activity for primary viruses, which in some cases can neutralize viruses of multiple clades. However, these antibodies are produced in relatively low titers, and require the presence of potent immunological adjuvants, which have not yet been approved for widespread human use. Recent studies have demonstrated that the immunogenicity of a number of peptides derived from the V3 region of HIV-1 gp120 or the region of gp41 bearing the 2F5 neutralizing epitope is greatly enhanced in BCG- sensitized animals and humans upon coupling to PPD. We will therefore prepared PPD-conjugated V1/V2 and V3 fusion glycoproteins to test as immunogens in several animals models. The immunogenicity of these proteins will be tested in BCG-primed and unprimed animals. The antigen specificity and neutralizing activities of the resulting antibodies will be tested against a number of virus isolates, including clinical isolates. In related experiments the V1/V2 and V3 fusion glycoproteins will be used to titer, isolate and characterize specific antibodies against these domains in sera of patients immunized with PPD-conjugated V3 peptides described in Project 1. The potential synergistic neutralizing effects of anti- V1/V2 and anti-V3 antibodies induced by these various immunogens will be assayed in vitro, and the potential enhancing effects of combining various V1/V2 and V3 PPD-conjugated immunogens on the neutralizing responses in immunized animals will also be measured. Finally, we will provide a series of monoclonal and polyclonal antibodies with potent HIV-neutralizing activities for evaluation in the engineered mouse models being developed in this program by Dr. Goldstein.